Utilizing micro-CT to evaluate bone structure surrounding · PDF filepentobarbitalnatrium...

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Utilizing micro-CT to evaluate bone structure surrounding dental implants: a comparison with histomorphometry Stefan Vandeweghe 1,2, Paulo G. Coelho 3, Christian Vanhove 4, Ann Wennerberg 1, Ryo Jimbo 1 1. Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö, Sweden. 2. Department of Periodontology and Oral Implantology, Dental School, Faculty of Medicine and Health sciences, University of Ghent, Belgium. 3. Department of Biomaterials and Biomimetics, New York University, New York, USA 4. Medical Imaging and Signal Processing, Faculty of Engineering and Architecture, University of Ghent, Belgium. Correspondence to: Dr. Stefan Vandeweghe, Department of Periodontology and Oral Implantology, University Hospital Ghent-P8. De Pintelaan 185, B-9000 Ghent, Belgium. Tel.: +32 9 332 40 17 Fax.: +32 9 332 35 51 E-mail: [email protected] Running title: Validity of micro-CT to evaluate bone around dental implants Key words: micro-ct, histomorphometry, dental implants, bone-implant-contact, osseointegration

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Utilizing micro-CT to evaluate bone structure surrounding dental implants: a

comparison with histomorphometry

Stefan Vandeweghe 1,2, Paulo G. Coelho 3, Christian Vanhove 4, Ann Wennerberg

1, Ryo Jimbo 1

1. Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö,

Sweden.

2. Department of Periodontology and Oral Implantology, Dental School, Faculty of

Medicine and Health sciences, University of Ghent, Belgium.

3. Department of Biomaterials and Biomimetics, New York University, New York,

USA

4. Medical Imaging and Signal Processing, Faculty of Engineering and Architecture,

University of Ghent, Belgium.

Correspondence to: Dr. Stefan Vandeweghe,

Department of Periodontology and Oral Implantology, University Hospital Ghent-P8.

De Pintelaan 185, B-9000 Ghent, Belgium.

Tel.: +32 9 332 40 17

Fax.: +32 9 332 35 51

E-mail: [email protected]

Running title: Validity of micro-CT to evaluate bone around dental implants

Key words: micro-ct, histomorphometry, dental implants, bone-implant-contact,

osseointegration

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ABSTRACT

Introduction

Although histology has proven to be a reliable method to evaluate the

ossoeintegration of a dental implant, it is costly, time consuming, destructive, and

limited to 1 or few sections. Micro-CT (µCT) is fast and delivers 3D information, but

this technique has not been widely used and validated for histomorphmetric

parameters yet. This study compared µCT and histomorphometry by means of

evaluating their accuracy in determining the bone response to 2 different implant

materials.

Materials and methods

Thirty-two titanium (Ti) and 16 hydroxyapatite (HA) implants were installed in 16 lop-

eared rabbits. After 2 and 4 weeks, the animals were sacrified and the samples

retrieved. After embedding, the samples were scanned with µCT, and analysed 3-

dimensionally for bone area (BA) and bone-implant-contact (BIC). Thereafter, all

samples were sectioned and stained for histomorphometry.

Results

For the Ti implants, the mean BIC was 25.25% and 28.86% after 2 and 4 weeks,

respectively, when measured by histomorphometry, while it was 24.11% and 24.53%

when measured with µCT. BA was 35.4% and 31.97% after 2 and 4 weeks for

histomorphometry and 29.06% and 27.65% for µCT. For the HA implants, the mean

BIC was 28.49% and 42.51% after 2 and 4 weeks, respectively, when measured by

histomorphometry, while it was 33.74% and 42.19% when measured with µCT. BA

was 30.59% and 47.17% after 2 and 4 weeks for histomorphometry and 37.16% and

44.95% for µCT.Direct comparison showed that only the 2 weeks BA for the titanium

implants was significantly different between µCT and histology (P=0.008).

Conclusion

Although the technique has its limitations, µCT corresponded well with

histomorphometry and should be considered as a tool to evaluate bone structure

around implants.

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INTRODUCTION

The success of osseointegration has brought in numerous benefits to the patients

needing long-lasting rehabilitation of one’s lost dentition 1-3. While successful from the

beginning of its practice, the quest for improvements in both short- and long-term

host-to-implant response has resulted in substantial evolution of implant fixture

design in the macro, micro, and nano surface levels. Then, these design changes are

often tested in laboratory animal models for performance evaluation by

histomorphometry or biomechanical testing.

However, as implant modifications become detailed and complex and studies

become multifactorial in nature in an attempt to develop an informed platform for

implant design optimization, it is sometimes difficult to capture detailed bone

response in a timely manner and with enough resolution with the currently

conventional methods. For example, the biomechanical removal torque testing may

not be able to resolve small differences in biomechanical performance, caused by

modifications of the implant surfaces at the nano level. Even histology, which has

been proven to provide us with both qualitative and quantitative information, has a

shortcoming due to the fact that it is based on one or a reduced number of sections

through the sample cut, which is a limited subset of the entire specimen. Other key

factors such as time to prepare the samples, destructiveness of the method, and cost

must be taken into consideration when performing these studies. Thus, new

evaluation techniques that increase evaluation throughput while providing at least the

same resolution ability as current techniques are highly desirable.

Currently, one of the potential candidate tools as an adjunct for biomechanical testing

is micro-computed-tomography (µCT), first described by Feldkamp et al. in the end of

the 1980’s4, has been used in the past for quantification and characterization of bone

architecture in relation to bone disorders. While clinical CT scanners typically

produce images composed of 1 mm3 volume elements (voxels), µCT produces

improved resolution in the range of a few µm, or approximately 1,000,000 times

smaller than regular CT scanning. It allows a non-destructive, 3D evaluation of the

specimen in high resolution within a limited amount of time, and also provides 3D

reconstructed images to obtain better understanding of bone architecture taking

place within the region of interest. Several studies have validated the technique of

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µCT by comparing it to histological sections of bone specimens and found a high

correlation between both techniques 5-7. However, one must keep in mind that the

samples in these studies consisted only from trabecular bone, and did not have

present metallic implantable devices in bone with the potential to metallic halation

and beam hardening. These artifacts are created by dense objects and depend on

the attenuation coefficient and geometry of the object 8. Naturally, this make

visualization of the bone within a 200 µm radius around the implant a challenge and

thereby evaluation at the bone-implant interface difficult 9. From a quantitative bone

morphometric perspective, the ultimate goal in the future would be skipping the time

consuming and costly histological section preparation while also decreasing the

number of samples and/or subjects necessary for both biomechanical and

morphometric components of the study. Ideally, the same sample providing bone

morphometry should also be mechanically tested. However, in order to move towards

this direction, it is important to prove the accuracy of 3D µCT based bone

morphometry even at the bone-implant interface region. In this study, 3D µCT data

was compared to 2D histology of titanium and hydroxyapatite implants, which were

placed in the rabbit bone.

The aim of this study was to examen the validity of micro-CT as a tool for evaluation

of implant osseointegration, by comparing 3-dimensional data with 2-dimensional

histomorphometry, and to evaluate the effect of material density and geometry on

beam hardening and micro-CT image quality.

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MATERIALS AND METHODS

Implants and implant surgery

Two implant materials were used for the study, namely titanium (Ti) and

hydroxyapatite (HA). For the Ti implants, thirty-six turned, commercially pure titanium

(Grade 4) threaded implants of 8 mm length and 3.3 mm diameter were used. For the

HA implants, sixteen polished non-threaded HA implants of length 9.0 mm and 4.2

mm diameter were used. The mass attenuation coefficient for titanium and

hydroxyapatite at 55keV is 0.99 cm2/g and 0.497 cm2/g, respectively.

Thereafter, the implants were placed unicortically in the condyle of the distal femur

(Ti) and proximal tibia (HA) of sixteen lop-eared rabbits (mean body weight, 3.9 kg).

This study was approved by the Malmö/Lund regional animal ethics committee

(approval number: M282-09).

Before surgery, the legs were shaved and disinfected with 70% ethanol and 70%

chlorhexidine. The animals were anesthetized with intramuscular injections of a

mixture of 0.15 mL/kg medetomidine (1 mg/mL Dormitor; Orion Pharma, Sollentuna,

Sweden) and 0.35 mL/kg ketamine hydrochloride (50 mg/mL Ketalar; Pfizer AB,

Sollentuna, Sweden). Lidocaine hydrochloride (Xylocaine; AstraZeneca AB,

Södertälje, Sweden) was administrated as the local anesthetic at each insertion site

at a dose of 1 mL. The implants were inserted with W&H implant unit (Elcomed, W&H

SA-310, Burmoos Austria) at a rotation speed of 20 revolutions/minute.

Postoperatively, buprenorphine hydrochloride (0.5 mL Temgesic; Reckitt Benckiser,

Slough, UK) was administered as an analgesic for three days.

After 2 and 4 weeks, the rabbits were sacrificed with an overdose (60 mg/mL) of

pentobarbitalnatrium (Apoteksbolaget AB, Stockholm, Sweden). The retrieved

samples were placed in 4 % formaldehyde for 24 hours and thereafter stored in 70 %

ethanol.

Micro Computed Tomography and 3D Reconstruction

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The 3D bone formations around the implants were examined using micro computed

tomography (μCT 40, Scanco Medical, Basserdorf, Germany) with a slice resolution

of 36 μm. Five hundred μCT slices were imaged at an X-ray energy level of 55 kV,

and a current of 145 μA. Integration time was 200 ms with a total scanning time of

36.3 min (128 mAs).

All data were exported in Dicom-format and imported in Amira (Visage Imaging

GmbH, Berlin, Germany) for evaluation. Since part of the implant was outside of the

bone, the data were cropped along the implant axis to where the cortical bone

started. Before segmentation, threshold levels for bone and implant were determined,

based on visual inspection of the complete slices and on the gray-scale histogram

(Figure 1). This was done by determining the upper and lower threshold levels for

bone and implant in 5 samples for each implant type. Threshold levels of bone and

implant did not overlap and allowed to make a clear distinction. The means were

calculated and used for every sample. Threshold determination was repeated for

intra-and inter-examiner repeatability evaluation.

Then, a region of interest (ROI) was defined, where the bone area (BA) would be

calculated. Therefore, the implant was selected based on its threshold level and this

region was circumferentially expanded, creating a 0.75 mm zone around the implant.

Subsequently, implant and bone in the ROI was differentiated based on their

threshold levels, creating 3 volumes: implant, bone and soft tissue/empty spaces.

These were converted into tetrahedral grid from triangular surfaces, to make area

and volume measurements possible. Outcome variables were Bone Area (BA), being

the percentage of bone that is present in the region around the implant and BIC

(Bone Implant Contact), being the area-percentage of the total implant surface that is

covered by bone.

Ground Section Preparation and Histological Analysis

After the μCT analysis, all samples were processed for undecalcified ground

sectioning. In brief, after a series of dehydration and infiltration in resin, the samples

were embedded in light-curing resin (Technovit 7200 VLC; Heraeus Kulzer

Wehrheim, Germany). Thereafter, one central undecalcified cut and ground section

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was prepared from each implant with the Exakt sawing and grinding equipment. The

sections were ground to a final thickness of about 40 µm and stained with toluidine

blue.

Histological evaluations were performed using a light microscope (Eclipse ME600,

Nikon, Japan) and histomorphometrical data was analyzed by an image analysis

software (Image J ver.1.43u; National Institutes of Health). Bone-Implant contact

(BIC) percentage and Bone Area (BA) percentage were calculated along the entire

implant and calculated with ×10 objective magnification and the amount of bone area

in the same defined area performed in the 3D analysis was calculated.

Statistical Analysis

The statistical analyses was done using SPSS software (SPSS Inc., Chicago, IL,

USA). Shapiro-Wilk tests for normaility were not statistically significant. Non-

parametric Wilcoxon signed rank test and Mann-Whitney U-Test with the significance

level set at 0.05 were used for statistical comparison. Pearsson test was used to

identify possible correlations between both methods.

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RESULTS

Overall

In total, 48 implants were installed (32 titanium, 16 hydroxyapatite). Routine clinical

inspections showed that healing after surgery progressed uneventfully, and there

were no clinical signs of infection. At the time of sacrifice, all implants were already

immobile suggesting osseointegration.

Histomorphometry

The histological sections presented newly formed trabeculae with deeply stained

mineralized tissue after 2 and 4 weeks of healing.

Ti implants

The mean BIC after 2 weeks was 25.25% (SD 7.22; range 15.14 – 36.81), while the

mean BA was 35.14% (SD 7.09; range 21.57 – 49.76). After 4 weeks, the mean BIC

was 28.86% (SD 8.73; range 14.99 – 51.41) and the mean BA 31.97% (SD 10.48;

range 14.71 – 50.55). There were no significant differences between both time

periods in BIC (p=0.397) or BA (p=0.198) (Figure 2).

HA implants

The mean BIC after 2 weeks was 28.49% (SD 17.15; range 3.03 – 50.43), while the

mean BA was 30.59% (SD 10.30; range 15.33 – 44.37). After 4 weeks, the mean BIC

was 42.51% (SD 9.45; range 26.69 – 54.18) and the mean BA 47.17% (SD 14.81;

range 28.28 – 71.50). There was no significant difference in BIC (p=0.074) between 2

and 4 weeks, but BA (p=0.036) increased significantly over time (Figure 3).

Micro-CT

The mean lower and upper threshold gray-levels for bone were 2902 (SD 187, range

2697 – 3135) and 7726 (SD 211, range 7566 – 8096). The threshold value for the

titanium implant was 12881. The mean lower and upper threshold level for the HA

implant were 8975 (SD 742, range 8203-9876) and 15480 (SD 568, range 14839-

15937).

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The intra-examiner repeatability on threshold level was high (90 % agreement within

5% deviation; Pearsson correlation coefficient: 0.998 - p<0.001; Wilcoxon signed

ranks test: p=0.139), as was the inter-examiner reproducibility (80 % agreement

within 5% deviation; Pearsson correlation coefficient: 0.997 - p<0.001; Wilcoxon

signed ranks test: p=0.059)

Ti implants

After 2 weeks, mean BIC was 24.11% (SD 6.93; range 10.22 – 37.74) and mean BA

29.06% (SD 6.53; range 19.64 – 40.18). After 4 weeks, the mean BIC was 24.53%

(SD 5.63; range 11.54 – 32.59), while the mean BA was 27.65% (SD 4.36; range

18.07 – 35.06). There were no significant changes in BIC (p=0.826) and BA

(p=0.397) between both time points (Figure 2).

HA implants

After 2 weeks, mean BIC was 33.74% (SD 8.75; range 25.57 – 52.37) and mean BA

37.16% (SD 9.18; range 26.53 – 55.63). After 4 weeks, the mean BIC was 42.19%

(SD 14.46; range 25.66 – 62.77), while the mean BA was 44.95% (SD 14.94; range

27.25 – 67.59). There were no significant changes in BIC (p=0.172) and BA

(p=0.294) between both time points (Figure 3).

Micro-CT versus Histomorphometry

Micro-CT analyses and histomorphometry corresponded well (Figure 4 and Figure 5)

and showed no significant differences at 2 weeks BIC (p=0.594), 4 weeks BIC

(p=0.085) and 4 weeks BA (P=0.058) for the titanium implants. However, the 2 weeks

BA was significantly different between µCT and histology (p=0.008). Both methods

showed a non-significant increase in BIC and a non-significant decrease in BA

between 2 and 4 weeks.

For the HA implants, no significant differences were observed in 2 weeks BIC

(p=0.327), 2 weeks BA (p=0.093), 4 weeks BIC (p=0.779) and 4 weeks BA (p=0.208)

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between histology and micro-CT. Histology showed a non-significant increase in BA

over time, howver micro-CT did not.

Overall, there was a significant correlation for BIC (p<0.001) as well as for BA

(p<0.001) between µCT and histomorphometry.

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DISCUSSION

Micro-CT is fast, non-destructive and allows 3D-evaluation compared to histological

sections. Therefore, this technology has been devoted significant interest over the

last years. In this study, we evaluated the accuracy of this technique by comparing it

with the histomorphometric results of the same samples, since very few studies have

validated the use of µCT as a technique to evaluate bone structures around implants.

Although the absolute values for µCT were slightly different from histology, there was

only a significant difference in the BA at 2 weeks for the titanium implants and

histomorphometry revealed a significant increase in BA over time around the HA

implants, while µCT did not. Possible differences between micro-CT and

histomorphometry may depend on the cutting direction and slice thickness of the

histological sections, as can be derived from the 3D images (Figure 6). As Sarve et

al.10 recently demonstrated, the results may vary with 30% depending on the cutting

direction. On the other hand, the micro-CT data may be affected by artifacts, such as

beam hardening.

Micro-CT allows evaluation of the total circumferential space while histomorphometry

is limited to 1 or few slices. Therefore, one could assume that micro-CT is more

accurate as the entire volume of the implant in bone sample is evaluated. in fact, a

few studies have evaluated this method in relation to BIC and BA. Van Oosteryck et

al.11 concluded that micro-CT and histomorphometry were very alike, although this

was based on an optical comparison of 1 sample. Similar to our study, Stoppie et

al.12 compared both techniques and concluded that the bone trabeculae were clearly

visible, but became difficult to detect in proximity of the implant with µCT. These 2

studies, however, compared 2D µCT with 2D histomorphometry. Although there was

a clear correlation between both methods, the authors reported that the results may

vary depending on the treshold level, voxel size and cutting direction of the scan, and

on the slice thickness and direction of the histological slide. Freilich et al.13 compared

bone height around 7 implants placed in rabbits by means of 3D µCT and

histomorphometry, and found a similar outcome.

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According to our results and also the literature in agreement, µCT appears to have a

strong potential to become an alternative for histological examination. However,

concerns have been expressed by multiple research groups. For instance, Schouten

et al.14 reported high discrepancy between µCT and histomorphometry regarding the

bone volume around the implant. According to the authors, this was due to the noise

around the implant and the threshold selection, which resulted in an overestimation

of the µCT values. Also, several other authors described a blurred border around the

implant due to metal artifacts caused by beam hardening, scatter and noise 12,14-16.

As this affects the voxels in immediate proximity around the implant, the size of the

blurred zone will depend on the resolution of the image and can therefore easily

range from 6 to 60µm. Therefore, it can be difficult to detect the bone structures in

this area. Determining the treshold levels based on the total image facilitates the

detection of the bony structures and might partially solve the problem.

Artifacts will also be influenced by the settings of the µCT and changes in energy-

level may improve results As Van Oosterwyck et al. 11 pointed out, smaller bone

trabeculae become invisible when the energy level is to high. In their study 90 and

130 kV were used, while the samples in our study were scanned at 55Kv. In

addition, higher voltages will also induce more artifacts.

Although no filters were used in our study, some studies recommend the use of an

aluminum filter11,12 That might explain why some streak artifacts were present, although it

did not interfere with the tresholding and quantification. Although some advocate

additional software filtering to improve the image quality, we chose not to use any

smoothening filters since this might change the result 17. Of course, we could not

exclude possible minor smoothening in the software’s algorithm that was used to

create the mesh that was used for quantification.

In the present study, 2 different materials were used, titanium and hydroxyapatite,

respectively. The mass attenuation coefficient for hydroxyapite and titanium at 55keV

is 0,497 cm2/g and 0.99 cm2/g, respectively. As a result, the X-rays will have different

interactions with these two different materials. The higher density of titanium can

result in a more blurred border around the implant due to beam hardening, scatter

and noise, which can affect the measurement of BIC and BA. Secondly, the geometry

of the two implants was different. The hydroxyapite inserts were cork shaped, while

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the titanium inserts contained a screw-thread. As a result of the more complex

structure of the titanium inserts, the delineation of the three volumes (implant, bone

and soft tissue/empty spaces), necessary to calculate BIC and BA, will be more

challenging. Figure 7 represents a colored version of the µCT slices, with each color

representing a different attenuation level. While the HA implant demonstrates almost

no halation, some halation can be seen around the TI implant, especially at the apical

part. This was also confirmed by Stoppie et al.12 and is probably related to the

geometry of the implant apex.

It is, however, difficult to determine the precise effect of material composition on

beam hardening, artifact creation and its effect on the measurements and extensive

experimentation for appropriate set ups should be performed for each equipment.

Although two different biomaterials were used, this study was not intended to

compare both implants. However, in a dog model, Mouzin et al.18 reported a

significantly higher BIC for loaded HA implants compared to titanium implants, which

is similar to the results in this study.

Although histology is still regarded as the “gold standard”, this technique also has

limitations. For instance, cutting and grinding the implant in bone blocks may damage

the interface and influence results. Also, Johansson & Morberg19 showed that thicker

slices results in an overestimation of measurements and therefore, sections over

30µm should be avoided. The biggest limitation, however, is that histology only

presents 2D information. Therefore, one may question if one or few slices will

accurately represent the total sample. Micro-CT will contain a lot more information

about the bone structures and may therefore be a more accurate representation. On

the other hand, µCT will have its limitations regarding the evaluation of bone

pathologic conditions 20. Whereas traditional histology will also deliver information

regarding cells, µCT will not. Therefore, µCT will never fully replace

histomorphometry. To a certain extent, however, it may serve as an alternative or

addition to histology or as a high throughput tool for bone morphometry in industrial

research and development scenarios.

CONCLUSION

Micro-CT corresponded well with histomorphometry and can therefore be a valuable

tool to examine bone structures around implants in a 3-dimensional way. Implant

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materal and geometry may affect halation and image quality, but do not necessarely

prevent peri-implant bone analysis.

ACKNOWLEDGEMENTS

Christian Vanhove is supported by the GROUP-ID consortium of Ghent University.

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FIGURES

Figure 1: Bone and implant were both defined as a region of interest, based on their threshold values. The yellow line marks a region of 0.75mm around the implant where the BA was measured. The data were cropped, leaving a region starting from the cortical bone to the apex of the implant (rectangular zone) in which the measurements were done. This procedure was similar for the titanium (a) and hydroxyapatite implants (b).

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Figure 2: Boxplot representing the BIC and BA for the titanium implants, obtained by

histomorphometry and micro-ct

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Figure 3: Boxplot representing the BIC and BA for the hydroxyapatite implants, obtained by histomorphometry and micro-ct.

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Figure 4: Figure representing the histological section (right) and the comparable layer from the micro-Ct image (left) for a Ti implant.

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Figure 5: Figure representing the histological section (right) and the corresponding layer of the micro-CT image (Left) for a HA implant.

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Figure 6: 3D representation of the titanium (left) and HA (right) implants in relation to the surrounding bone.

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Figure 7: Color transformation of CT slices of a (A) hydroxyapatite and (B) titanium implant. The different color represent different attenuation levels. Almost no halation can be seen around the HA implant, while some halation can be seen around the Ti implant (light green), especially around the apical part.

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